Pluripotent stem cell differentiation

Abstract

The present invention relates to the field of pluripotent stem cell differentiation. The present invention provides methods for the differentiation of pluripotent stem cells on a human feeder cell layer. In particular, the present invention provides an improved method for the differentiation of pluripotent stem cells into pancreatic endocrine cells using a human feeder cell layer.

1 . A method for differentiating pluripotent stem cells, comprising the steps of:
 a. Culturing the pluripotent stem cells,   b. Plating the pluripotent stem cells onto a human feeder cell layer, and   c. Treating the pluripotent stem cells with at least one factor that promotes the differentiation of the pluripotent stem cells.   
   
   
       2 . The method of  claim 1 , wherein the human feeder cell layer comprises dermal fibroblast cells. 
   
   
       3 . The method of  claim 1 , wherein the human feeder cell layer comprises foreskin fibroblast cells. 
   
   
       4 . The method of  claim 1 , wherein the human feeder cell layer comprises pancreatic-derived stromal cells. 
   
   
       5 . The method of  claim 1 , wherein the pluripotent stem cells are embryonic stem cells. 
   
   
       6 . The method of  claim 5 , wherein the embryonic stem cells are human embryonic stem cells. 
   
   
       7 . The method of  claim 4 , wherein the pancreatic-derived stromal cells are substantially negative in the expression of at least one protein marker selected from the group consisting of NCAM, ABCG2, cytokeratin 7, cytokeratin 8, cytokeratin 18, and cytokeratin 19. 
   
   
       8 . The method of  claim 4 , wherein the pancreatic-derived stromal cells are substantially positive in the expression of at least one protein marker selected from the group consisting of CD44, CD73, CD90 and CD105. 
   
   
       9 . The method of  claim 1 , wherein the pluripotent stem cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage. 
   
   
       10 . The method of  claim 1 , wherein the pluripotent stem cells are differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage. 
   
   
       11 . The method of  claim 1 , wherein the pluripotent stem cells are differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage. 
   
   
       12 . The method of  claim 1 , wherein the step of culturing the pluripotent stem cells is accomplished on an extracellular matrix. 
   
   
       13 . The method of  claim 1 , wherein the step of culturing the pluripotent stem cells is accomplished on a human feeder cell layer. 
   
   
       14 . A method to treat diabetes, comprising the steps of:
 a. Culturing the pluripotent stem cells,   b. Plating the pluripotent stem cells onto a human feeder cell layer,   c. Treating the pluripotent stem cells with at least one factor that promotes the differentiation of the pluripotent stem cells, and   d. Transplanting pluripotent stem cells that have been treated with at least one factor that promotes the differentiation of pluripotent stem cells into a human patient having diabetes.   
   
   
       15 . The method of  claim 14 , wherein the step of treating the pluripotent stem cells with at least one factor that promotes the differentiation of the pluripotent stem cells causes the pluripotent stem cells to differentiate into cells of the pancreatic endocrine lineage. 
   
   
       16 . The method of  claim 15 , wherein the differentiation of the pluripotent stem cells is accomplished in two or more steps, each step requiring the treatment of cells by at least one factor that promotes the differentiation of cells into one or more of definitive endoderm, pancreatic endoderm, and pancreatic endocrine lineage.